Expression in Escherichia coli of UDP-glucose pyrophosphorylase cDNA from potato tuber and functional assessment of the five lysyl residues located at the substrate-binding site

The entire structural gene for potato tuber UDP-glucose pyrophosphorylase has been amplified from its cDNA by the polymerase chain reaction and inserted into the expression plasmid pTV118-N downstream from the lac promoter. Escherichia coli JM105 cells carrying thus constructed plasmid produced the enzyme to a level of about 5% of the total soluble protein upon induction with isopropyl beta-D-thiogalactopyranoside. The recombinant enzyme purified to homogeneity in two column chromatographic steps was structurally and catalytically identical with the enzyme purified from potato tuber except for the absence of an N-terminal-blocking acetyl group. To examine functional roles of the five lysyl residues that had been identified by affinity labeling studies to be located at or near the active site of the enzyme [Kazuta, Y., Omura, Y., Tagaya, M., Nakano, K., & Fukui, T. (1991) Biochemistry (preceding paper in this issue)], they were replaced individually by glutamine via site-directed mutagenesis. The Lys-367----Gln mutant enzyme was almost completely inactive, and the Lys-263----Gln mutant enzyme had significantly decreased Vmax values with perturbed Km values for pyrophosphate and alpha-D-glucose 1-phosphate. Lys-329----Gln also exhibited increased Km values for these substrates but exhibited Vmax values similar to those of the wild-type enzyme. The two mutant enzymes Lys-409----Gln and Lys-410----Gln showed catalytic properties almost identical with those of the wild-type enzyme. Thus, among the five lysyl residues, Lys-367 is essential for catalytic activity of the enzyme and Lys-263 and Lys-329 may participate in binding of pyrophosphate and/or alpha-D-glucose 1-phosphate.     

Enzymatic synthesis of arbutin undecylenic acid ester and its inhibitory effect on melanin synthesis

Transesterification of arbutin and undecylenic acid vinyl ester was catalyzed by alkaline protease, Bioprase, in dimethylformamide to get arbutin derivative having undecylenic acid at 6-position of glucose moiety, 6-O-undecylenoyl p-hydroxyphenyl beta-D-glucopyranoside. The reaction rate increased with increase of arbutin concentration, and when its concentration was 0.9 M, the conversion rate was more than 90% under addition of 2 M undecylenic acid vinyl ester. The obtained arbutin ester significantly suppressed melanin production in murine B16 melanoma cells.     

HIV-1-derived self-inactivating lentivirus vector induces megakaryocyte lineage-specific gene expression

Pluripotent, self-renewing, hematopoietic stem cells are considered good targets for gene modification to treat a wide variety of disorders. However, as many genes are expressed in a stage-specific manner during the course of hematopoietic development, it is necessary to establish a lineage-specific gene expression system to ensure the proper expression of transduced genes in hematopoietic stem cells. In this study, we constructed a VSV-G-pseudotyped, human immunodeficiency virus type 1-based, self-inactivating lentivirus vector that expressed green fluorescent protein (GFP) under the control of the human CD41 (glycoprotein 2b; GP2b) promoter; this activity is restricted to megakaryocytic lineage cells. The recombinant virus was used to infect human peripheral blood CD34+ (hematopoietic stem/progenitor) cells, and lineage-specific gene expression was monitored with GFP measurements. The analysis by FACS determined that GFP expression driven by the GP2b promoter was restricted to megakaryocytic progenitors and was not present in erythrocytes. Furthermore, in the hematopoietic colony-forming assay, GFP expression was restricted to colony-forming units-megakaryocyte (CFU-Meg) colonies under the control of the GP2b promoter, whereas all myeloid colonies (burst-forming units-erythroid, colony-forming units-granulocyte-macrophage, and CFU-Meg) expressed GFP when the transgene was regulated by the cytomegalovirus promoter. These results demonstrated lineage-specific expression after gene transduction of hematopoietic stem cells. The application of this vector system should provide a useful tool for gene therapy to treat disorders associated with megakaryocyte (platelet) dysfunction.     

Renal defects associated with improper polarization of the CRB and DLG polarity complexes in MALS-3 knockout mice

Kidney development and physiology require polarization of epithelia that line renal tubules. Genetic studies show that polarization of invertebrate epithelia requires the crumbs, partition-defective-3, and discs large complexes. These evolutionarily conserved protein complexes occur in mammalian kidney; however, their role in renal development remains poorly defined. Here, we find that mice lacking the small PDZ protein mammalian LIN-7c (MALS-3) have hypomorphic, cystic, and fibrotic kidneys. Proteomic analysis defines MALS-3 as the only known core component of both the crumbs and discs large cell polarity complexes. MALS-3 mediates stable assembly of the crumbs tight junction complex and the discs large basolateral complex, and these complexes are disrupted in renal epithelia from MALS-3 knockout mice. Interestingly, MALS-3 controls apico-basal polarity preferentially in epithelia derived from metanephric mesenchyme, and defects in kidney architecture owe solely to MALS expression in these epithelia. These studies demonstrate that defects in epithelial cell polarization can cause cystic and fibrotic renal disease.     

Elemental analysis of lung tissue particles and intracellular iron content of alveolar macrophages in pulmonary alveolar proteinosis

Background

Pulmonary alveolar proteinosis (PAP) is a rare disease occurred by idiopathic (autoimmune) or secondary to particle inhalation. The in-air microparticle induced X-ray emission (in-air micro-PIXE) system performs elemental analysis of materials by irradiation with a proton microbeam, and allows visualization of the spatial distribution and quantitation of various elements with very low background noise. The aim of this study was to assess the secondary PAP due to inhalation of harmful particles by employing in-air micro-PIXE analysis for particles and intracellular iron in parafin-embedded lung tissue specimens obtained from a PAP patient comparing with normal lung tissue from a non-PAP patient. The iron inside alveolar macrophages was stained with Berlin blue, and its distribution was compared with that on micro-PIXE images.

Results

The elements composing particles and their locations in the PAP specimens could be identified by in-air micro-PIXE analysis, with magnesium (Mg), aluminum (Al), silicon (Si), phosphorus (P), sulfur (S), scandium (Sc), potassium (K), calcium (Ca), titanium (Ti), chromium (Cr), copper (Cu), manganase (Mn), iron (Fe), and zinc (Zn) being detected. Si was the major component of the particles. Serial sections stained by Berlin blue revealed accumulation of sideromacrophages that had phagocytosed the particles. The intracellular iron content of alveolar macrophage from the surfactant-rich area in PAP was higher than normal lung tissue in control lung by both in-air micro-PIXE analysis and Berlin blue staining.

Conclusion

The present study demonstrated the efficacy of in-air micro-PIXE for analyzing the distribution and composition of lung particles. The intracellular iron content of single cells was determined by simultaneous two-dimensional and elemental analysis of paraffin-embedded lung tissue sections. The results suggest that secondary PAP is associated with exposure to inhaled particles and accumulation of iron in alveolar macrophages.     

New thiazole derivatives as potent and selective 5-hydroxytriptamine 3 (5-HT3) receptor agonists for the treatment of constipation

The syntheses and biological evaluation of a series of novel indeno[1,2-d]thiazole derivatives are described. Several groups reported 5-HT(3) receptor agonists which were mainly evaluated for their activities on the von Bezold-Jarisch reflex (B-J reflex). We discovered that tetrahydrothiazolopyridine derivative 1b had a contractile effect on the isolated guinea pig colon with weak B-J reflex. Our efforts to find a new type of 5-HT(3) receptor agonists on the isolated guinea pig colon focused on the synthesis of a fused thiazole derivative 1d modified from 1b and reverse-fused thiazole derivatives (7-10). In this series, 10f (YM-31636) showed high affinity and selectivity for the cloned human 5-HT(3) receptor; furthermore, it showed potent and selective 5-HT(3) receptor agonistic activity. YM-31636 was examined for its effects on defecation in animals, thus evaluating the compound as an agent against constipation.